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Total IRS2 cellular kit HTRF®

The total IRS2 assay enables the detection of cellular IRS2 and can be used as a normalization assay for the phospho-IRS2 kit.
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  • Ready-to-use Ready-to-use
  • High sensitivity High sensitivity
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
The total IRS2 assay enables the detection of cellular IRS2 and can be used as a normalization assay for the phospho-IRS2 kit.
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Overview

Insulin receptor substrates (IRS) 1 and 2 are the primary substrates of Insulin/IGF1 receptors, and mediate cell growth/survival and metabolic homeostasis. While IRS1 is primarily found in adipose tissue, IRS2 is the predominant form in pancreatic-beta cells and the central nervous system (CNS). Decreased IRS2 levels are associated with diabetes and neurodegeneration, whereas highly overexpressed levels of IRS2 drive tumor motility and invasion. This cell-based assay measures the modulation of IRS2 protein levels.

Benefits

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  • PRECISION

Total IRS2 assay principle

The total IRS2 assay quantifies the expression level of IRS2 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-IRS2 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of IRS2 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.
Total IRAK3 Assay principle

Total IRS2 2-plate Assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding total IRS2 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total IRAK3 2-plate Assay protocol

Total IRS2 1-plate assay protocol

Detection of total IRS2 with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

IRS2 upregulation in INS-1E beta rat cells using glucose and forskolin

INS-1E cells were plated in a 24-well plate and cultured for 4 days. A starvation step using Krebs Ringer Buffer (KRB) without glucose was performed for 1 hour before stimulating the cells with glucose +/- forskolin (diluted in culture medium without glucose). After 6 hours of treatment, the medium was removed and cells were lysed and transferred into a 384-well low volume white microplate with HTRF total IRS2 detection reagents added. The HTRF signal was recorded after an overnight incubation.
IRS2 upregulation in INS-1E beta cells using glucose
Upregulation of IRS2 protein levels in INS-1E rat pancreatic ß-cells using glucose and forskolin

Upregulation of IRS2 protein levels in HEK293 human embryonic kidney cells using forskolin

HEK293 cells were plated at 100,000 cells/well in a 96-well plate, and incubated for 24 hours at 37°C, 5% CO2. After treatment for 30 minutess or 4 hours with 1 nM forskolin, the medium was removed and cells were lysed with 50 µL of supplemented lysis buffer #1 for 30 minutess at RT under gentle shaking. 16 µL of lysate were then transferred into a 384-well low volume white microplate and 4 µL of the HTRF total IRS2 detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Upregulation of IRS2 in HEK293 cells using forskolin

Detection of IRS2 in SH-SY5Y human neuroblastoma cell line

SH-SY5Y cells were plated in a 6-well plate at two different cell densities (2 million and 4 million cells/well) and incubated for 24 hours at 37°C, 5% CO2. The medium was then removed and cells were lysed with 200 µL of supplemented lysis buffer #1 for 30 minutess at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF total IRS2 detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Detection of IRS2 in SH-SY5Y human neuroblastoma cell line

Detection of IRS2 in MCF-7 human breast cancer cell line

MCF-7 cells were plated in a 96-well plate at 100,000 cells/well and incubated for 24 hours at 37°C, 5% CO2. The medium was then removed and cells were lysed with 50 µL of supplemented lysis buffer #1 for 30 minutess at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF total IRS2 detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Detection of IRS2 in MCF-7 human breast cancer cell line

Function and regulation of IRS2 in pancreatic ß-cells

IRS2 expression in pancreatic-beta cells by both 2nd messengers: glucose and GLP-1. These promote activation of transcription factor CERB leading to increased IRS2 expression. IRS2 is crucial to functional Insulin/IGF-1 signaling, promotiing beta-cell growth, survival and function. Pro-inflammatory adipocytokines, free fatty acids and cellular stress promote IRS2 degradation and contribute to spontaneous beta-cell apoptosis, decreased beta-cell mass, insulin deficiency and eventually metabolic disorders such as diabetes.
IRS2 signaling pathway in pancreatic beta cells

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

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Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

Product Insert IRS2 total Kit / 64IRTPEG-64IRTPEH

64IRTPEG-64IRTPEH - Product Insert

HTRF Product Catalog 2020 July update

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

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